Diagnosis of intestinal parasitic disease is confirmed by recovery and identification of helminth eggs and larvae or protozoan trophozoites and/or cysts in fecal samples. One of the problems faced by the parasitologist is the delay between collection and examination of specimens. Since trophozoites deteriorate quickly outside of the host, it has been difficult in the past to deliver or ship fecal samples to offsite laboratories for accurate diagnosis or to hold the sample for diagnosis at a later time. Even though organisms are present in specimens when first obtained from the patient, they my be unrecognizable or completely disintegrated by the time they are processed, leading to inaccurate diagnosis.
Investigators have emphasized the importance of proper collection of samples for the diagnosis of infections by helminths, especially protozoa. Unless samples are examined immediately after defecation, or are preserved in a suitable fixative, many infections may be missed due to the short lives of the protozoan trophozoites (Yang et al. 67 Amer. J. Clin. Pathol. 300 (1977)).
To overcome the problems associated with the examination of fecal samples, a number of fixatives have been proposed for the preservation of trophozoites and other diagnostic stages in recognizable form. These include fixatives whose composition comprises compounds such as formaldehyde, formalin, mercury, phenol, and polyvinyl alcohol (PVA). However, the use of these types of preservatives results in a number of disadvantages.
The use of formaldehyde, mercury and phenol containing preservatives raises environmental and health concerns. Vapors of formaldehyde and phenol are intensely irritating to mucous membranes, and topical application may produce acute dermatitis. Further, mercury compounds are readily absorbed via the respiratory tract, intact skin and gastrointestinal tract, and can contribute to physiological problems including neurological disorders. Mercury has also been known to bioaccumulate in the environment, resulting in environmental problems.
In addition to the health and environmental problems associated with formaldehyde and mercury based preservatives, the use of these type of preservatives limits the procedures used to analyze the preserved sample. A commonly used diagnostic method utilizes a two-vial kit for collecting stool specimens (Brooke, 4 Triangle 326 (1960)). In accordance with the two-vial method, the person collecting the specimen is directed to introduce portions of the specimen into a vial containing a formalin solution, and into a second vial containing PVA fixative. Hence, the two-vial method requires the collection of two separate fecal samples in order to provide a complete analysis.
Subsequent processing of specimens taken using the two-vial method result in undesirable process steps. For example, the formalin fixed sample is concentrated with filtration, centrifugation and partitioning with an organic solvent, such as ether or ethyl acetate. The sample is utilized for the detection of cysts, eggs and larvae. The second specimen, which was fixed in PVA, is spread on a microscope slide, stained and examined for cysts and trophozoites. Hence, the processing of formaldehyde or formalin fixed specimens requires concentration steps which utilize organic solvents and which can result in the loss of parasites from the sample (see Examples).
The use of formaldehyde or formalin as a fixative also results in limitations on the types of staining and detection techniques that can be subsequently utilized. For example, the use of specific antibodies to detect particular cell types is commonly used in histological applications. Antibodies can recognize and bind to specific receptors on a cell. The bound antibody is rendered detectable through techniques which include fluorescent or radioactive labels. The chemical action of formaldehyde tends to alter receptive sites on cells and thus render them undetectable with antibody. Hence, the use of a formaldehyde based fixative precludes the use of potentially useful immunoassay methods.
Attempts have been made to alleviate some of the problems associated with previous methods for fixing and analyzing fecal samples for parasites. For example, the Para-Pak.TM. product (sold by Meridian Diagnostics, Inc. Cincinnati, Ohio) provides a multipurpose fixative-preservative which allows for the recovery and identification of all stages of intestinal parasites. However, the fixative contains formaldehyde which results in all of the aforementioned disadvantages.
Formaldehyde substitutes have also been described. For example, U.S. Pat. No. 3,546,334 to Lerner et al. describes a cytological fixative and protective composition that includes a liquid solution of alcohol, water, polyethylene glycol, acetone and optionally a dye. The .composition is applied directly to a smear of cells on a microscope slide to provide a method for simultaneously fixing the specimen on the slide and protecting it with a coating. The '334 patent does not suggest that its composition could be useful for fixing fecal specimens and makes no suggestion that its claimed composition would be useful for fixing and subsequent examination of samples containing parasites.
Formalin replacements which are diagnostically equivalent to formaldehyde but are 100% free of formaldehyde and glutaraldehyde also available (sold as NoToX.TM. Histological Fixative, Earth Safe Industries, Inc. Belle Mead, New Jersey). The formalin substitute is described as a unique bis carbonyl compound and as being useful for the preservation of histological samples. Its use for the preservation of fecal samples is not described or suggested. Further, the NoToX.TM. formalin replacement had an unacceptably high pH, did not provide clear nuclear definition of cysts, and was found to be unacceptable for the fixation-and subsequent analysis of fecal samples for parasites.
In addition to the problems associated with formaldehyde preservatives, conventional techniques for applying fixed sample material to a microscope slide and subsequent staining procedures often can result in loss of sample from the slide. In accordance with commonly accepted practice, specimens which have been fixed in PVA, and formalin fixed samples which have been concentrated are applied as a smear to a microscope slide. In some methods, albumin is mixed with the specimen prior to its application to the slide (Technical Literature for Para-Pak.TM. Fixative, Meridian Diagnostics, Inc. Cincinnati, Ohio). Smears are usually allowed to air dry and are then stained through a process which includes subjecting the smear to a series of staining and washing steps with organic solvents and aqueous solutions. During the staining and washing steps, sample can be lost from the slide, resulting in the need for repeat analysis or inaccurate diagnosis.
An object of this invention is to provide a fixative composition which does not contain formaldehyde, formalin, mercury or other harmful chemicals commonly associated with fixative compositions.
Another objective of this invention is to provide a method of using the fixative composition in a process for fixing, concentrating and staining fecal specimens being analyzed for ova or parasites such that only one fecal specimen is needed for both concentrating and staining fecal specimens being analyzed for ova or parasites.
Another important objective of this invention is to provide a method which does not require an organic solvent extraction step and does not result in the loss of parasites during concentration of the sample.
Another objective of this invention is to provide a method of fixing fecal samples which does not preclude the use of immunoassay techniques.
Yet another objective of this invention is to provide method that utilizes a slide fixative composition that results in improved adhesion of sample to a microscope slide.
Further objects and advantages of the invention will be found by reference to the following description.